It is well established that IFN plays an important role in antiviral immunity. Our cells are equipped with an extensive network of innate immune sensing mechanisms for detecting invading pathogens through recognition by PRRs. When a PRR is engaged, it triggers a signaling pathway that often leads to the activation of IFN expression3
. Infection with enveloped viruses also trigger an IFN-independent pathway that involves the direct activation by IRF3 of a subset of ISGs28
. In fact, IRF3 can bind promoters of many ISGs in addition to IFN genes41
. Promoters of IFN genes are complex (e.g. Ifnb1
), containing both positive and negative regulatory elements for IRFs, NF-κB and AP-1, and a concerted effort of multiple transcription factors is often required for their stimulation. In contrast, promoters of many ISGs are simpler (e.g. Ifit1
), and can be easily turned on by IRFs independently of IFN9,41
. Direct activation of antiviral genes is important for nonprofessional IFN-producing cells such as fibroblasts to effectively defend themselves against viral infection, or for cells to defend themselves against viruses that have evolved mechanisms to disrupt the IFN response. It is also advantageous for cells to rapidly induce some ISGs upon viral infection before a stronger and more sustained response can be established by IFN signaling pathways. A recent study of the cytosolic RNA sensing pathway provided strong evidence that IFN-independent activation of ISGs mediated by peroxisomal MAVS is functionally important for defense against RNA virus infections10
Very little is known about whether IFN-independent activation of ISGs occurs in the absence of infection, and how it is regulated. Here, we identified Trex1, a cytosolic protein associated with the ER, as a key negative regulator of IFN-independent activation of Ifit1
and other ISGs in uninfected cells. When the function of Trex1 is disrupted, either by genetic knockout in mice, or by a homozygous mutation in humans, or by siRNA knockdown in a variety of cell types, a subset of ISGs were activated independently of IFN, leading to an antiviral state. Remarkably, the ISG induction in Trex1-deficient cells is sustained at very high amounts and achieves an antiviral state that is comparable to that caused by the IFN-dependent pathway. This is in contrast to the viral infection induced IFN-independent response in WT cells that appears to be temporary and less robust10
. We have also challenged WT and Trex1−/−
mouse cells or TREX1R114H/R114H
human cells with a variety of RNA viruses including VSV, influenza, Sendai and West Nile virus; and they all failed to replicate in cells that have lost Trex1 function.
We have also identified an innate immune pathway, involving STING-TBK1-IRF3-IRF7 that is important for the IFN-independent ISG activation in Trex1-deficient cells. STING is a critical adaptor protein for sensing pathogen-associated DNA or cyclic di-GMP in the cytosol and subsequent induction of IFN expression2,7
. Our data expands the function of the STING-TBK1-IRF3-IRF7 pathway to include both IFN-dependent and independent branches as downstream pathways. A recent study also showed that STING activates STAT6 phosphorylation upon viral infection, which then induces chemokines such as CCL2, CCL20 and CCL26 and immune cell homing8
. We did not observe induction of these chemokines in Trex1−/−
cells compared to WT (data not shown). Together, STING, and associated innate immune factors are becoming a versatile machinery that can activate multiple distinct downstream pathways.
Our data also shed some light on the potential endogenous trigger of IFN-independent ISG activation. We found that Trex1-deficient or mutant cells contain excessive amount of lysosomal vacuoles and expanded lysosomal compartments as determined by immunofluroscence and immunoblot analysis of lysosomal markers, quantitative RT-PCR analysis of lysosomal genes and by electron microscopy. Consistent with elevated lysosome biogenesis, the master regulator of lysosome genes, TFEB translocates to become predominantly nuclear in Trex1−/−
cells. We also found that mTORC1 activity is reduced in Trex1−/−
cells and is restored after Flag-Trex1 expression in Trex1−/−
cells, suggesting that Trex1 plays an important role in maintaining mTORC1 activity, which regulates TFEB nuclear translocation35,39
. We also provided several lines of evidence to demonstrate that TFEB-regulated lysosomal biogenesis is functionally linked to ISG activation: TFEB knockdown in Trex1 deficient cells tempered ISG activation and antiviral immunity; TFEB overexpression in WT cells, which promotes lysosomal biogenesis32
, increased Ifit1
expression; chloroquine treatment of WT cells, which induces nuclear translocation of TFEB35
and exhibits antiviral activity36–38
, increased Ifit1
expression up to 15-fold; and mTOR knockdown by siRNA in WT cells also increased Ifit1
expression. Furthermore, based on our observation of elevated lysosomal genes and protein expression and lack of excess accumulation of undigested contents, Trex1−/−
cells are likely to have enhanced lysosomal function. One could imagine that the release of abnormally high amounts of processed peptide or nucleic acids into the cytosol, or into the extracellular space (via exocytosis42
), might break cellular homeostasis or immune tolerance or exceed the threshold for cytosolic DNA sensing. The exact identity of these cytosolic DNA remains unclear, and previous studies have indicated DNA replication debris20
and endogenous retroelements19
. Aberrant functions of lysosomes have been indicated in lupus nephritis where lysosomal contents mimic viral particles and activate innate immunity43
. It is also possible that increased lysosome vacuoles could cause membrane perturbation that would elicit IFN-dependent or -independent antiviral response9,44
. Further studies are required to distinguish these possibilities. Together, our work demonstrate an important link between lysosomal biogenesis and innate immune activation of ISGs, as well as a novel role for TREX1 in regulating lysosomal biogenesis through TFEB and mTORC1.
We have previously shown that Trex1 inhibits HIV-mediated IFN activation11
. Here, we validated this finding, and further uncovered a novel function of Trex1 in the regulation of IFN-independent innate immune activation through lysosomal biogenesis in uninfected cells, which results in a broad-spectrum antiviral state in which the replication of several different RNA viruses is inhibited. Both functions of Trex1 share a similar innate immune signaling pathway that involves STING-TBK1-IRF3, which can activate multiple downstream pathways. The upstream stimuli for HIV-mediated IFN activation is HIV DNA from nonproductive reverse transcription11
, whereas the upstream stimuli for the IFN-independent pathway likely involves lysosome function.
Our work also provides further insight into pathogenetic mechanisms underlying systemic autoimmunity associated with TREX1
mutation such as SLE, a prototypic autoimmune disease. Central to SLE pathogenesis is that ineffective waste disposal due to impaired apoptosis or defective clearance of cellular debris leads to excessive release of autoantigens which activate innate immune sensors and trigger immune responses leading to formation of autoantibodies45
. Our findings unravel a novel mechanism for a cell-intrinsic mechanism of initiation of autoimmunity due to enhanced lysosome function. Moreover, the constitutive type I IFN-independent ‘ISG-signature’ which is detectable in a variety of cell types and tissues may potentially represent a valuable biomarker that could be applied as clinical outcome measure.
In summary, our study uncovered a signaling cascade that involves the biogenesis of a cellular organelle (e.g. lysosome) and cytosolic innate immune detection. Both segments of the cascade function together to establish an antiviral state in Trex1-deficient cells independently of IFN activation or viral infection. We identified many components of this cascade, some of which (e.g. TFEB and mTORC1) have not been directly implicated in intrinsic antiviral immunity. We have also uncovered novel functions of known innate immune regulators such as Trex1 and STING. Further understanding of the mechanism by which this signaling cascade is regulated will have important implications not only for understanding antiviral defense but also pathogenic mechanisms underlying autoimmune diseases.